The invention generally relates to confocal microscopes and more particularly to programmable spatially light modulated or programmable array microscopes and to a microscopy method which employs freely programmable patterns for illumination and/or detection.
Confocal microscopy based on point scanning systems with conjugate pairs of illumination and detection apertures is an effective tool for imaging a microscopic object to be investigated with direct optical sectioning. The discrete aperture spots are illuminated in the object plane of the microscope from which reflected or fluorescent light is observed through the conjugate detection apertures in an image plane. Commonly used confocal microscopes based on scanning systems with mechanically translated aperture disks (so-called Nipkow disks with a plurality of apertures) or with rotating mirrors being adapted to scan an object with a laser beam (Confocal Laser Scanning Microscopy, CSLM).
Both scanning systems have certain limitations. The aperture disks yield particularly a restriction of the illumination field, a degraded contrast and high intensity losses. Typically less than 3% of the disk is transmissive since the spacing between the pinholes must be large to maintain the confocal effect. On the other hand, the scanning mirrors of CSLM result in a low duty cycle imposed by the sequential single point data acquisition.
The problem of intensity losses has been countered by the introduction of aperture correlation microscopes as described by R. Ju{haeck over (s)}kaitis, T. Wilson et al. in xe2x80x9cNaturexe2x80x9d (vol. 383, 1996, p. 804-806) and by T. Wilson, R. Ju{haeck over (s)}kaitis et al. in xe2x80x9cOptics Lettersxe2x80x9d (vol. 21, 1996, p. 1879-1881). Such a microscope as schematically shown in FIG. 9 uses for specimen illumination a multiple-point source being formed by a combination of the light source with a programmable aperture mask. The detection of the light reflected by the specimen is obtained by a camera through the same aperture mask. The aperture mask is a fast spatial light modulator formed by an array of addressable pixels or a rotating disk with fixedly impressed modulation codes.
The mask carries a pattern of uncorrelated openings and closings increasing the transmissivity of the disk to about 50%. Due to the correlation avoiding coding sequence used by Ju{haeck over (s)}kaitis et al., the detected image is a superposition of a confocal image with a conventional image. For obtaining a final confocal image, it is necessary to detect independently a separate conventional image (e.g. by a blank sector in a rotating disk) to be subtracted from said superposition.
This additional detection of a conventional image is time consuming, so that only a restricted data acquisition rate is available. The applicability of the aperture correlation technique is further limited due to the restricted transmissivity. Therefore, fluorescence measurements are only possible in exceptional cases with high fluorescence yields. A corresponding increase of the illumination intensity could lead to unacceptable photodamage or bleaching reactions.
With regard to these disadvantages, an improved spatially light modulated microscope has been described by M. Liang et al. in xe2x80x9cOptics Lettersxe2x80x9d (vol. 22, 1997, p. 751-753) and in the corresponding U.S. Pat. No. 5,587,832. This prior art microscope is schematically shown in FIG. 10. A two-dimensional spatial light modulator is formed by a digital micromirror device (in the following: xe2x80x9cDMDxe2x80x9d) which reflects the illumination light from the source (laser or white light source) to the probe and the detection light from the probe to a two-dimensional detector. Each micromirror of the DMD is individually controllable to form an illumination and detection spot or not.
The use of a DMD as a light modulator allows the direct detection of confocal images. Furthermore it is possible to determine the minimum confocal pattern period (distance of micromirror forming illuminating spots) without compromising the confocality. Nevertheless, the microscope of U.S. Pat. No. 5,587,832 suffers from a limited illumination intensity as only a part of the object reflected light can be used for imaging. Furthermore, this light used for imaging has an xe2x80x9cout-of-focusxe2x80x9d offset influencing the SNR of the confocal image in an disadvantageous manner. Finally, the prior art microscope is specialized to confocal imaging without the possibility of obtaining conventional images.
The real-time confocal microscopy or imaging, in particular in the field of imaging biological objects like cells or parts thereof, calls for further improvements with regard to sensitivity, detection speed and for an extended applicability by the implementation of further measurement principles.
It is the object of the present invention to provide an improved device and method for confocal imaging allowing rapid data acquisition, in particular with effective optical sectioning, high spatial resolution and/or high optical efficiency. It is a particular object of the invention to provide rapid two- or three-dimensional imaging of biological or chemical materials (e.g. living cells, reaction composites etc.) and thus information about molecular structure and function. Due to the inherent sensitivity and selectivity, molecular fluorescence is a preferred spectroscopic phenomenon to be implemented with the new imaging device and method.
The above object is solved by a confocal imaging device or method comprising the features of claim 1 or 14, respectively. Advantageous embodiments of the invention are defined in the dependent claims.
The basic idea of the inventors is the operation of a confocal optical imaging system as e.g. a programmable spatially light modulated confocal microscope (in the following: PAM) with spatial light modulator means (in the following: SLM) such that the entire light output from a specimen or object is dissected into two images being collected simultaneously or subsequently. Generally, spatial light modulator means comprise an array of modulator elements the transmission or reflections properties of which being individually controllable. With a first group of SLM modulator elements (xe2x80x9conxe2x80x9d-elements), the object is illuminated and a focal conjugate image is collected, while with a second group of SLM modulator elements (xe2x80x9coffxe2x80x9d-elements), a non-conjugate image is collected. The non-conjugate image contains out-of-focus light. The illumination spots formed by the SLM modulator elements are focused to a focal plane of the object.
When positioned at the image plane of a microscope, the SLM elements each of which being conjugate to a distinct point in the focal plane of the object define a programmable array which is used for illumination and/or detection.
The modulator elements of the first group are individually controllable such that the pattern sequence of illumination spots is represented by a time-dependent systematically shifting grid pattern or a pattern based on a pseudo-random sequence of finite length. In the first case, the first image is an image corresponding to a confocal image and the second image is a difference image between a non-confocal image and the first image. In the second case, the first image is a superposition of confocal image and a non-confocal image and the second image is a difference image between a non-confocal image and a confocal image. In any case, the first image can contain a portion of a conjugate image as shown in FIG. 2.
The SLM can be operated in transmission or reflection mode. Depending on the number of detector systems of the detection means, the PAM can be built as a so-called single path or double path PAM. According to a preferred arrangement, the light source means of the confocal optical imaging system contain a white light lamp and means for wavelength selection.
Preferred applications of the confocal optical imaging system are investigations in the cell and tissue biology, analytical/biotechnological procedures, in-situ hybridization in genetic analysis, formation of an optical mask for position selective photochemical reactions, generation and readout of biochemical arrays on chips, large scale surface inspections in the semiconductor industry, and/or optical recording and read-out.
The invention has the following advantages. The PAM can be implemented as a modular extension of existing microscopes. The out-of-focus light normally rejected with conventional CLSM is collected and used to enhance the in-focus image. Each element of the first group of modulator elements individually is controllable or programmable such that the pattern sequence of illumination spots illuminates at least one predetermined region-of interest of the object. The implementation of a highly flexible white light microscope capable of full field and/or confocal imaging (with laser as option) is available. Due to a high optical throughput, a white light illumination is allowed yielding an extended applicability of the microscope.
The simultaneous use of both images delivered via the xe2x80x9conxe2x80x9d-elements and the xe2x80x9coffxe2x80x9d-elements is a unique feature of the invention allowing in particular improved deconvolution algorithms.